The goals of this project are to elucidate the molecular mechanisms of receptor activation and intracellular signaling that are initiated by the pressor octapeptide, angiotensin II (Ang II). Most of the diverse physiological actions of Ang II in cardiovascular, renal, neuronal, and other target cells are mediated by the Gq/11 protein-coupled AT1 receptor. The functions of the distantly related AT2 receptor in general counteract the growth-related actions of the AT1 receptor. The C-terminal region of the third intracellular loop of the AT1 angiotensin receptor is an important determinant of G protein coupling. A mutational analysis of the roles of individual residues in Gq/11-dependent phosphoinositide signaling identified Ile(238) and Phe(239) as the critical residues in this region of the third loop for G protein activation. It is likely that a conserved apolar amino acid corresponding to Ile(238) of the AT(1)-R is a general requirement for signaling by other G protein-coupled receptors. Together with the earlier identification of the Tyr(215) and Leu(223) residues in the N-terminal region of the third intracellular loop, these studies have demonstrated that four specific amino acids are required for Gq/11 coupling to this domain of the AT1 receptor, and probably of other GPCRs. The angiotensin AT2 receptor is an unusual seven transmembrane domain glycoprotein that is coupled to activation of tyrosine phosphatase and inhibition of MAPK kinase, and does not undergo agonist-induced internalization. An investigation of the potential role of receptor phosphorylation in AT2 function revealed that Ang II caused rapid and specific phosphorylation of a single residue (Ser354) located in the cytoplasmic tail of the receptor. This was prevented by the AT2 antagonist, PD123177, and by inhibition of PKC. In cells co-expressing AT1 and AT2 receptors, Ang II-induced phosphorylation of the AT2 receptor was reduced by either PD123177 or the AT1 receptor antagonist, DuP753, and was abolished by treatment with both antagonists or with PKC inhibitors. These findings indicate that the AT2 receptor is rapidly phosphorylated via PKC during homologous activation by Ang II, and also undergoes heterologous PKC-dependent phosphorylation during activation by the AT1 receptor. The latter process could contribute to activation of the counter-regulatory action of AT2 receptors on AT1 receptor-mediated growth responses. In most GPCRs, agonist-induced phosphorylation at serine/threonine residues is predominantly mediated by specific receptor kinases (GRKs), and is dependent on adjacent acidic residues located in the C-terminal tail or the third intracellular loop of the receptor. No such residues were found in the AT1 receptor tail. However, an analysis of the functional role of a diacidic motif (Asp236-Asp237) in its third intracellular loop revealed that substitution of both amino acids with alanine (DD-AA) or asparagine (DD-NN) residues diminished Ang II-induced AT1 receptor phosphorylation in COS-7 cells. However, the ability of the phosphorylation-deficient receptor to mediate Ang II-stimulated inositol phosphate production, mitogen-activated protein kinase (MAP kinase) activation, and AT1 receptor desensitization and internalization, was not significantly impaired. Overexpression of dominant negative GRK2(K220M) decreased agonist-induced receptor phosphorylation by ~40%, but did not further reduce the impaired phosphorylation of DD-AA and DD-NN receptors. Inhibition of protein kinase C (PKC) by bisindolylmaleimide reduced the phosphorylation of both the wild type and the DD-mutant receptors by ~30%. The inhibitory effects of GRK2(K220M) expression and PKC inhibition on agonist-induced phosphorylation were additive for the wild-type AT1-R, but not for the DD-mutant receptor. Agonist-induced internalization of the wild type and DD-mutant receptors was similar and was unaltered by coexpression of dominant negative GRK2. These findings have demonstrated that an acidic motif located in the third intracellular loop of the AT1-R is required for agonist-induced phosphorylation of its carboxyl-terminal tail by GRKs. In addition, the properties of the DD-mutant receptor suggest that not only Ang II-induced signaling, but also receptor desensitization and internalization are independent of GRK-mediated phosphorylation of the agonist-activated AT1 receptor.